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Overexpression of phosphomimic mutated OsWRKY53 leads to enhanced blast resistance in rice.

Chujo T, Miyamoto K, Ogawa S, Masuda Y, Shimizu T, Kishi-Kaboshi M, Takahashi A, Nishizawa Y, Minami E, Nojiri H, Yamane H, Okada K - PLoS ONE (2014)

Bottom Line: WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses.An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster).When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

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Post-translational regulation of OsWRKY53 transactivation activity by OsMKK4DD.A, Diagrams of effector, reporter, and reference plasmids used in transient reporter gene assays. B, Regulation of transactivation activity of OsWRKY53 by OsMKK4DD in cultured rice cells. A GUS construct was used as a negative control. Firefly LUC activity was normalized against that of Renilla LUC. Values of LUC activity are shown relative to those of GAL4-W53 + GUS (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.01 by One-way ANOVA with Tukey post hoc test). C, Enhanced transactivation of a phosphorylation-mimic mutant of OsWRKY53 in cultured rice cells. Transient reporter gene assay was performed as in B. Values of LUC activity are shown relative to those of GAL4-W53 (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test). D, Expression analysis of GAL4DB and GAL4DB-OsWRKY53 variants in cultured rice cells. qRT-PCR analysis was performed using total RNA isolated from rice cells after particle bombardment with effector, reporter and reference plasmids. Values indicate relative mRNA levels normalized to the expression of the RLUC gene (n = 4); bars indicate the standard error of the mean. W53, W53SA and W53SD indicate the native OsWRKY53, an OsWRKY53 mutants whose Ser residues in the SP cluster were substituted to Ala, and an OsWRKY53 mutant that mimics the phosphorylated form, respectively.
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pone-0098737-g003: Post-translational regulation of OsWRKY53 transactivation activity by OsMKK4DD.A, Diagrams of effector, reporter, and reference plasmids used in transient reporter gene assays. B, Regulation of transactivation activity of OsWRKY53 by OsMKK4DD in cultured rice cells. A GUS construct was used as a negative control. Firefly LUC activity was normalized against that of Renilla LUC. Values of LUC activity are shown relative to those of GAL4-W53 + GUS (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.01 by One-way ANOVA with Tukey post hoc test). C, Enhanced transactivation of a phosphorylation-mimic mutant of OsWRKY53 in cultured rice cells. Transient reporter gene assay was performed as in B. Values of LUC activity are shown relative to those of GAL4-W53 (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test). D, Expression analysis of GAL4DB and GAL4DB-OsWRKY53 variants in cultured rice cells. qRT-PCR analysis was performed using total RNA isolated from rice cells after particle bombardment with effector, reporter and reference plasmids. Values indicate relative mRNA levels normalized to the expression of the RLUC gene (n = 4); bars indicate the standard error of the mean. W53, W53SA and W53SD indicate the native OsWRKY53, an OsWRKY53 mutants whose Ser residues in the SP cluster were substituted to Ala, and an OsWRKY53 mutant that mimics the phosphorylated form, respectively.

Mentions: It has also been reported that N. benthamiana MEK2DD enhanced transactivation activity of NbWRKY8 [29]. To investigate whether the rice OsMKK4DD correspondingly enhances transactivation activity of OsWRKY53, we performed a transient reporter gene assay. We constructed effector plasmids that contained a GUS or OsMKK4DD gene under the control of the maize ubiquitin promoter (Fig. 3A). We also constructed effector plasmids that contained the CaMV 35S promoter, driving a gene that encodes a fusion protein of the DNA-binding domain of the yeast transcriptional activator GAL4 and the full-length OsWRKY53 or OsWRKY53SA (GAL4DB-OsWRKY53 or GAL4DB-OsWRKY53SA), and a control plasmid encoding only GAL4DB (Fig. 3A). Each of the GAL4DB-OsWRKY53-variant effector plasmids or the control plasmid was delivered into rice cells along with a reporter plasmid GAL4-TATA-LUC-NOS, which contained 5 tandem repeats of a GAL4 binding site fused to the firefly LUC (FLUC), and either GUS or OsMKK4DD effector plasmid by particle bombardment. As described in our previous report [36], coexpression of GAL4DB-OsWRKY53 with GUS showed more than 20-fold greater LUC activity compared to coexpression of GAL4DB with GUS as the negative control. On the other hand, LUC activity of rice cells coexpressing GAL4DB-OsWRKY53SA with GUS was reduced by 67% compared to those co-expressing GAL4DB-OsWRKY53 with GUS (Fig. 3B). Interestingly, coexpression of GAL4DB-OsWRKY53 with OsMKK4DD significantly increased (more than doubled) the LUC activity (Fig. 3B). In contrast, coexpression of GAL4DB-OsWRKY53SA with OsMKK4DD did not increase LUC activity (Fig. 3B). These results indicate that OsMKK4DD can increase transactivation activity of OsWRKY53 in an SP cluster-dependent manner.


Overexpression of phosphomimic mutated OsWRKY53 leads to enhanced blast resistance in rice.

Chujo T, Miyamoto K, Ogawa S, Masuda Y, Shimizu T, Kishi-Kaboshi M, Takahashi A, Nishizawa Y, Minami E, Nojiri H, Yamane H, Okada K - PLoS ONE (2014)

Post-translational regulation of OsWRKY53 transactivation activity by OsMKK4DD.A, Diagrams of effector, reporter, and reference plasmids used in transient reporter gene assays. B, Regulation of transactivation activity of OsWRKY53 by OsMKK4DD in cultured rice cells. A GUS construct was used as a negative control. Firefly LUC activity was normalized against that of Renilla LUC. Values of LUC activity are shown relative to those of GAL4-W53 + GUS (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.01 by One-way ANOVA with Tukey post hoc test). C, Enhanced transactivation of a phosphorylation-mimic mutant of OsWRKY53 in cultured rice cells. Transient reporter gene assay was performed as in B. Values of LUC activity are shown relative to those of GAL4-W53 (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test). D, Expression analysis of GAL4DB and GAL4DB-OsWRKY53 variants in cultured rice cells. qRT-PCR analysis was performed using total RNA isolated from rice cells after particle bombardment with effector, reporter and reference plasmids. Values indicate relative mRNA levels normalized to the expression of the RLUC gene (n = 4); bars indicate the standard error of the mean. W53, W53SA and W53SD indicate the native OsWRKY53, an OsWRKY53 mutants whose Ser residues in the SP cluster were substituted to Ala, and an OsWRKY53 mutant that mimics the phosphorylated form, respectively.
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pone-0098737-g003: Post-translational regulation of OsWRKY53 transactivation activity by OsMKK4DD.A, Diagrams of effector, reporter, and reference plasmids used in transient reporter gene assays. B, Regulation of transactivation activity of OsWRKY53 by OsMKK4DD in cultured rice cells. A GUS construct was used as a negative control. Firefly LUC activity was normalized against that of Renilla LUC. Values of LUC activity are shown relative to those of GAL4-W53 + GUS (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.01 by One-way ANOVA with Tukey post hoc test). C, Enhanced transactivation of a phosphorylation-mimic mutant of OsWRKY53 in cultured rice cells. Transient reporter gene assay was performed as in B. Values of LUC activity are shown relative to those of GAL4-W53 (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test). D, Expression analysis of GAL4DB and GAL4DB-OsWRKY53 variants in cultured rice cells. qRT-PCR analysis was performed using total RNA isolated from rice cells after particle bombardment with effector, reporter and reference plasmids. Values indicate relative mRNA levels normalized to the expression of the RLUC gene (n = 4); bars indicate the standard error of the mean. W53, W53SA and W53SD indicate the native OsWRKY53, an OsWRKY53 mutants whose Ser residues in the SP cluster were substituted to Ala, and an OsWRKY53 mutant that mimics the phosphorylated form, respectively.
Mentions: It has also been reported that N. benthamiana MEK2DD enhanced transactivation activity of NbWRKY8 [29]. To investigate whether the rice OsMKK4DD correspondingly enhances transactivation activity of OsWRKY53, we performed a transient reporter gene assay. We constructed effector plasmids that contained a GUS or OsMKK4DD gene under the control of the maize ubiquitin promoter (Fig. 3A). We also constructed effector plasmids that contained the CaMV 35S promoter, driving a gene that encodes a fusion protein of the DNA-binding domain of the yeast transcriptional activator GAL4 and the full-length OsWRKY53 or OsWRKY53SA (GAL4DB-OsWRKY53 or GAL4DB-OsWRKY53SA), and a control plasmid encoding only GAL4DB (Fig. 3A). Each of the GAL4DB-OsWRKY53-variant effector plasmids or the control plasmid was delivered into rice cells along with a reporter plasmid GAL4-TATA-LUC-NOS, which contained 5 tandem repeats of a GAL4 binding site fused to the firefly LUC (FLUC), and either GUS or OsMKK4DD effector plasmid by particle bombardment. As described in our previous report [36], coexpression of GAL4DB-OsWRKY53 with GUS showed more than 20-fold greater LUC activity compared to coexpression of GAL4DB with GUS as the negative control. On the other hand, LUC activity of rice cells coexpressing GAL4DB-OsWRKY53SA with GUS was reduced by 67% compared to those co-expressing GAL4DB-OsWRKY53 with GUS (Fig. 3B). Interestingly, coexpression of GAL4DB-OsWRKY53 with OsMKK4DD significantly increased (more than doubled) the LUC activity (Fig. 3B). In contrast, coexpression of GAL4DB-OsWRKY53SA with OsMKK4DD did not increase LUC activity (Fig. 3B). These results indicate that OsMKK4DD can increase transactivation activity of OsWRKY53 in an SP cluster-dependent manner.

Bottom Line: WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses.An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster).When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

Show MeSH
Related in: MedlinePlus